Apparatus and Methods for Transmitting Light

ABSTRACT

An apparatus and method for imaging includes an imaging system formed of a movable objective stage proximal to a sample and positioned for providing an excitation beam onto and for capturing an emission from the sample. The movable objective stage includes an optical lens apparatus and a turn reflector optically coupled to the imaging optics, where at least one of the optical lens apparatus and the turn reflector are movable relative to one another for scanning the sample, and wherein the movement is achieved while maintaining a substantially fixed optical path length between the optical lens apparatus and a fixed plane in a fixed imaging optics stage.

RELATED APPLICATION SECTION

This application claims the benefit of and priority to U.S. ProvisionalPatent Application No. 63/262,025, filed Oct. 1, 2021, and U.S.Provisional Patent Application No. 63/402,397, filed Aug. 30, 2022, thecontent of each which is incorporated by reference herein in theirentireties and for all purposes.

BACKGROUND

Imaging systems that scan samples, such as high throughput sequencerstations, rely upon movement of the samples relative to the imagerassembly or upon movement of the imager assembly relative to the sampleto achieve scanning. Such movement requires careful control andprecision of the movement and position of the movable components.However, depending on the application, moving the sample can beproblematic, especially with large flowcell cartridges having largenumbers of fluidic interfaces as these make sample movement relative tofixed optics substantially more challenging. Further, moving opticimagers can be problematic as these imagers are typically large devicesthat are prone to performance-degrading misalignment when subject overtime to numerous acceleration and deceleration events.

SUMMARY

Disadvantages of the prior art can be overcome and benefits as describedlater in this disclosure can be achieved through the provision ofapparatus and methods for transmitting light. Various implementations ofthe apparatus and methods are described below, and the apparatus andmethods, including and excluding the additional implementationsenumerated below, in any combination (provided these combinations arenot inconsistent), may overcome these shortcomings and achieve thebenefits described herein.

In accordance with a first implementation, an apparatus comprises orincludes: an imaging system having an excitation source for generatingan excitation beam, a fixed imaging optics stage composed of anexcitation source for generating an excitation beam, a sensor formeasuring an emission from a sample, and an imaging optics for imagingthe emission from the sample onto the sensor; and a movable objectivestage proximal to the sample and positioned for providing the excitationbeam onto the sample and for capturing the emission from the sample,where the movable objective stage includes an optical lens apparatus anda turn reflector optically coupled to the imaging optics of the fixedimaging optics stage, and where at least one of the optical lensapparatus and the turn reflector of the movable objective stage aremovable relative to one another for scanning of the sample, whilemaintaining a fixed optical path length between the optical lensapparatus and a fixed plane in the fixed imaging optics stage duringmovement.

In accordance with a second implementation, an apparatus comprises orincludes: an imaging system having an excitation source for generatingan excitation beam, a fixed imaging optics stage composed of a sensorfor measuring an emission from a sample, and imaging optics for imagingthe emission from the sample onto the sensor; and an objective stageproximal to the sample and positioned for providing the excitation beamonto the sample and for capturing the emission from the sample, wherethe objective stage includes an optical lens apparatus, wherein theimaging system comprises (i) one or more color separating elementsbetween the objective and the fixed imaging optics to direct light of afirst emission wavelength to a first image sensor of the sensor andlight of a second emission wavelength to a second image sensor of thesensor, or (ii) the one or more color separating elements within orafter the fixed imaging optics to direct light of the first emissionwavelength to the first image sensor and light of the second emissionwavelength to the second image sensor.

In accordance with a third implementation, a computer-implemented methodof optically probing a sample, the method comprises or includes:aligning, using one or more processors, a movable objective stage,having an optical lens apparatus and a turn reflector optically coupledto imaging optics of a fixed imaging optics stage, to align the opticallens apparatus with the sample for probing at an optical path length;providing, using the optical lens apparatus, an excitation beam to thesample and capturing, using the optical lens apparatus, a fluorescenceemission from the sample; in response to identification of a shift infocus at the sample from the fluorescence emission, adjusting a positionof the optical lens apparatus or a position of the turn reflector tocompensate for the shift; and moving, using the one or more processors,the optical lens apparatus and the turn reflector to position theoptical lens apparatus over a subsequent sample for probing, whilemaintaining the optical path length.

In accordance with a fourth implementation, an apparatus comprises orincludes: an excitation source, a movable objective stage, a movableimaging stage, a first actuator, a second actuator, and a controller.The excitation source is for generating a sampling beam. The movableobjective stage comprises or includes an objective. The objective stageis configured to receive the sampling beam from the excitation source,project the sampling beam onto a sample, and capture an emission fromthe sample resulting from the sampling beam. The movable imaging stagecomprises or includes an imaging sensor, and imaging optics for imagingthe emission from the sample onto the imaging sensor. The first actuatoris controllable to move the objective stage between different samplepositions and the second actuator is controllable to move the imagingstage. The controller is configured to control the first actuator andthe second actuator such that the imaging stage moves counter to theobjective stage to allow a length of an optical path between theobjective and the imaging sensor to remain substantially constant.

In accordance with a fifth implementation, a method, comprising orincluding controlling, using one or more processors, a first actuator tomove a movable objective stage by a first amount in a first direction tooptically align an objective of the objective stage with a sample at afirst sample position; controlling, using one or more processors, asecond actuator to move a movable imaging stage by the first amount in asecond direction opposite the first direction. The imaging stagecomprises or includes an imaging sensor, and moving the objective stageand the imaging stage by the first amount in opposite directionsmaintains a substantially constant optical path length between theobjective and the imaging sensor. The method also comprises or includesproviding a sampling beam to the objective stage. The objective stage isconfigured to project the sampling beam onto the sample. The method alsocomprises or includes imaging, using the objective stage and a pair ofturning mirrors, a fluorescence emission from the sample resulting fromthe sampling beam onto the imaging sensor.

In further accordance with the foregoing first, second, third, fourth,and/or fifth implementations, an apparatus and/or method may furthercomprise or include any one or more of the following:

In another implementation, the movable objective is movable in twoorthogonal directions to maintain a fixed optical path length.

In another implementation, the excitation source comprises or includes afirst excitation source producing a first excitation at a first samplingwavelength that elicits a first sample emission range of wavelengths anda second excitation source producing a second excitation at a secondsampling wavelength that elicits a second sample emission range ofwavelengths, each of the first excitation, first emission, secondexcitation and second emission having a respective optical path.

In another implementation, the apparatus further comprises or includes:a compensation plate positioned in one of the respective optical paths.

In another implementation, the apparatus further comprises or includes:a compensation plate positioned in a plurality of the respective opticalpaths.

In another implementation, both the optical lens apparatus and the turnreflector of the movable objective stage are movable relative to oneanother for scanning of a sample area.

In another implementation, at least one of the optical lens apparatusand the turn reflector of the movable objective stage are movablerelative to one another for scanning multiple samples areas at differentpositions.

In another implementation, the apparatus further comprises or includes:a controller configured to move the at least one of the optical lensapparatus and the turn reflector of the movable objective stage whilemaintaining the fixed optical path length to sample at the differentpositions.

In another implementation, the controller is configured to continuouslymove the optical lens apparatus and the turn reflector of the movableobjective between the different positions.

In another implementation, the apparatus further comprises or includes:a controller configured to continuously control movement of the turnreflector during capture of the emission beam from the sample tocompensate for vibrational effects during capture.

In another implementation, the apparatus further comprises or includes:a controller configured to continuously control movement of the opticallens apparatus and the turn reflector during capture of the emissionbeam from the sample to compensate for vibrational effects duringcapture.

In another implementation, the controller is configured to continuouslycontrol movement of the optical lens apparatus and the turn reflector atdifferent movement increments.

In another implementation, the apparatus further comprises or includes:a controller configured to move the movable objective to achieve thefixed optical path length at each of the different sample positions.

In another implementation, the apparatus further comprises or includes:a z-stage adjustment controller to adjust a distance between the opticallens apparatus and the sample.

In another implementation, the fixed imaging optics stage, the opticallens apparatus, and the turn reflector form a relay lens assembly forimaging the emission into the sensor.

In another implementation, the fixed imaging optics stage, the opticallens apparatus, and the turn reflector form an infinite conjugate lensassembly or near infinite conjugate lens assembly.

In another implementation, the fixed imaging optics stage and theoptical lens apparatus with the turn reflector each form a finiteconjugate lens assembly.

In another implementation, the apparatus further comprises or includes:one or more color separating elements between the objective and thefixed imaging optics to direct light of a first emission wavelength to afirst image sensor and light of a second emission wavelength to a secondimage sensor.

In another implementation, the movable objective stage is separatelymovable along two orthogonal axes each substantially planar to thesample.

In another implementation, the apparatus further comprises or includes:one or more color separating elements within or after the fixed imagingoptics to direct light of a first emission wavelength to a first imagesensor and light of a second emission wavelength to a second imagesensor

In another implementation, the apparatus further comprises or includes:a compensating plate disposed before a first image sensor.

In another implementation, the apparatus further comprises or includes:a plurality of compensating plates disposed before a first image sensor.

In another implementation, the apparatus further comprises or includes:a plurality of compensating plates disposed before a first image sensorand a different compensation plate or plurality of compensating platesis disposed before a second image sensor

In another implementation, one or more compensating plates is tilted orwedged.

In another implementation, the movable objective stage is separatelymovable along two orthogonal axes each substantially parallel to thesample.

In another implementation, the apparatus further comprises or includes acompensating plate disposed before a first image sensor.

In another implementation, the apparatus further comprises or includes aplurality of compensating plates disposed before a first image sensor.

In another implementation, the apparatus further comprises or includes aplurality of compensating plates disposed before a first image sensorand a different plurality of compensating plate disposed before a secondimage sensor.

In another implementation, the one or more compensating plates is(are)tilted or wedged.

In another implementation, the apparatus further comprises or includes acompensating plate pair disposed within a beam path defined by the oneor more color separating elements.

In another implementation, the compensating plate pair comprises a firstcompensating plate tilted in a first angular direction and a secondcompensating plate tilted in a second angular direction, equal andopposite to the first angular direction.

In another implementation, the one or more color separating elements aretilted about a first axis and the first compensating plate and thesecond compensating plate are each tilted about a second axis orthogonalto the first axis and to the optical axis.

In another implementation, the method further comprises or includesmoving, using the one or more processors, the optical lens apparatus andthe turn reflector to position the optical lens apparatus over thesubsequent sample for probing while maintaining the optical path lengththroughout the movement from the sample to the subsequent sample.

In another implementation, the method further comprises or includesperforming imaging processing on image data containing the fluorescenceemission; and in responding to determining the image data does notsatisfy a focusing condition, adjusting a vertical distance between theoptical lens apparatus and the sample until the image data satisfies thefocusing condition.

In another implementation, the method further comprises or includes,moving the optical lens apparatus and the turn reflector to position theoptical lens apparatus over the subsequent sample for probing, whilemaintaining the optical path length comprises moving the optical lensapparatus and the turn reflector in a plane substantially parallel to aplane containing the sample and the subsequent sample.

In accordance with an implementation, the apparatus comprises orincludes coupling optics positioned between the objective stage and theimaging stage along the optical path.

In accordance with another implementation, the coupling optics arefixed.

In accordance with another implementation, the coupling optics compriseor include a pair of turning mirrors positioned between the objectivestage and the imaging stage along the optical path.

In accordance with another implementation, the turning mirrors compriseor have faces positioned at approximately 45° angles.

In accordance with another implementation, the controller is configuredto cause the first actuator to move the objective stage toward thecoupling optics and cause the second actuator to move the imaging stageaway from the coupling optics.

In accordance with another implementation, the controller is configuredto cause the first actuator to move the objective stage away from thecoupling optics and cause the second actuator to move the imaging stagetoward the coupling optics.

In accordance with another implementation, the imaging optics of theimaging stage comprise or include relay optics.

In accordance with another implementation, the objective stage comprisesor includes imaging optics comprising or including relay optics.

In accordance with another implementation, the relay optics of theimaging stage and the relay optics of the objective stage reshape atleast one of the sampling beam or emission to compensate for spatialdispersion.

In accordance with another implementation, at least one of the firstactuator or the second actuator comprises or includes a drive motor, alinear motor, a voice coil motor, a ball screw, a stepper motor, or abelt drive.

In accordance with another implementation, the first actuator and thesecond actuator comprise or include a shaft comprising or having a firstthreaded portion and a second threaded portion, corresponding first andsecond ball nuts, and a motor to rotate the shaft. The imaging stagecarrying the first ball nut and the objective stage carrying the secondball nut.

In accordance with another implementation, the first threaded portioncomprises or has threads facing a first direction and the secondthreaded portion comprises or has threads facing a second directiondifferent from the first direction.

In accordance with another implementation, the motor rotates the shaftin a first direction and causes the first ball nut and the second ballnut to move toward one another and the motor rotates the shaft in asecond direction and causes the first ball nut and the second ball nutto move away from one another.

In accordance with another implementation, the objective stage furthercomprises or includes second coupling optics.

In accordance with another implementation, the coupling optics compriseor include a first pair of turning mirrors and the second couplingoptics comprise or include a second pair of turning mirrors.

In accordance with another implementation, one of the second pair ofturning mirrors redirects the sampling beam onto the sample.

In accordance with another implementation, the other of the second pairof turning mirrors redirects the emissions from the sample toward thefirst pair of turning mirrors.

In accordance with another implementation, the coupling optics compriseor include a pair of turning mirrors and the second coupling opticscomprise or include a second turning mirror.

In accordance with another implementation, the second turning mirrorredirects the sampling beam onto the sample.

In accordance with another implementation, the second turning mirrorredirects the emissions from the sample toward the first pair of turningmirrors.

In accordance with another implementation, the objective stage, thefirst actuator, the imaging stage, and the second actuator areconfigured and arranged such that a first center of mass of theobjective stage and a second center of mass of the imaging stage movealong substantially a same axis.

In accordance with another implementation, the objective stage, thefirst actuator, the imaging stage, and the second actuator areconfigured and arranged such that moving the objective stage and theimaging stage at a same time results in substantially no net forceapplied to the apparatus.

In accordance with another implementation, controlling the firstactuator comprises or includes controlling the first actuator to movethe objective stage towards a pair of turning mirrors, and controllingthe second actuator comprises or includes controlling the secondactuator to move the imaging stage away from the pair of turningmirrors.

In accordance with another implementation, the first actuator comprisesor includes controlling the first actuator to move the objective stagetowards a midline of the pair of turning mirrors, and controlling thesecond actuator comprises or includes controlling the second actuator tomove the imaging stage away from the midline of the pair of turningmirrors.

In accordance with another implementation, the first actuator and thesecond actuator comprise or include a shaft comprising or having a firstthreaded portion and a second threaded portion, corresponding first andsecond ball nuts, and a motor to rotate the shaft and controlling thefirst and second actuators comprises or includes controlling the motorto rotate the shaft such that the objective stage moves in the firstdirection, and the imaging stage moves in the second direction.

It should be appreciated that all combinations of the foregoing conceptsand additional concepts discussed in greater detail below (provided suchconcepts are not mutually inconsistent) are contemplated as being partof the subject matter disclosed herein and/or may be combined to achievethe particular benefits of a particular aspect. In particular, allcombinations of claimed subject matter appearing at the end of thisdisclosure are contemplated as being part of the subject matterdisclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a schematic diagram of an implementation of anoptical imager apparatus in accordance with the teachings of thisdisclosure, showing an imaging system with fixed imaging optics and amoveable objective stage.

FIGS. 2A-2E illustrate an objective lens apparatus and turn reflector indifferent positions while maintaining fixed the optical path length withan imaging in accordance with the teachings of this disclosure

FIGS. 3 and 4 illustrate a schematic of an objective lens apparatus anda turn reflector, both of a movable objective stage, in differentpositions, while maintaining fixed the optical path length with animaging in accordance with the teachings of this disclosure.

FIGS. 5-7 illustrate different positions of a movable objective stage inaccordance with the teachings of this disclosure.

FIG. 8 is a flow diagram of an example process for moving a movableobjective stage while maintaining a fixed optical path length inaccordance with the teachings of this disclosure.

FIG. 9 illustrates an example turn reflector having a compensation platefor compensating for multi-spectral emissions in accordance with theteachings of this disclosure.

FIG. 10 illustrates an example of compensation plate configuration forcorrecting for astigmatism in accordance with the teachings of thisdisclosure.

FIGS. 11A-11D illustrate an example turn reflector assembly havingcompensation plates for compensating for different induced spatiallyseparated beam paths between a fixed imaging optics and sensors inaccordance with teachings of this disclosure.

FIG. 12 illustrates a configuration of an apparatus having a movableobjective stage capable of moving in two translational directions inaccordance with the teachings of this disclosure.

FIG. 13 illustrates a schematic diagram of an implementation of a systemin accordance with the teachings of this disclosure.

FIG. 14 is a schematic illustration of an example imaging system thatcan be used to implement the disclosed implementations.

FIG. 15 is a top down view of a portion of an example imaging systemthat can be used to implement the imaging system of FIG. 14 .

FIG. 16 is a top down view of a portion of another example imagingsystem that can be used to implement the imaging system of FIG. 14 .

FIG. 17 is a side view of a portion of yet another example imagingsystem that can be used to implement the imaging system of FIG. 14 .

FIG. 18 is a side view of a portion of another example imaging systemthat can be used to implement the imaging system of FIG. 14 .

FIG. 19 is a flowchart representing an example method that can be usedto operate the imaging systems of FIGS. 14-18 or any of the disclosedimplementations.

FIG. 20 illustrates a schematic diagram of an implementation of a systemin accordance with the teachings of this disclosure.

DETAILED DESCRIPTION

Although the following text discloses a detailed description ofimplementations of methods, apparatuses and/or articles of manufacture,it should be understood that the legal scope of the property right isdefined by the words of the claims set forth at the end of this patent.Accordingly, the following detailed description is to be construed asexamples only and does not describe every possible implementation, asdescribing every possible implementation would be impractical, if notimpossible. Numerous alternative implementations could be implemented,using either current technology or technology developed after the filingdate of this patent. It is envisioned that such alternativeimplementations would still fall within the scope of the claims.

At least one aspect of this disclosure is directed to an apparatus andmethod for imaging. The apparatus may include an imaging system thateliminates the need to move either the sample or the entire imagerassembly itself, as with conventional techniques. Instead, the apparatusmay be designed to move only optics proximate to the sample, whilemaintaining the bulk of the imager assembly in a fixed position. Theimaging system can operate faster, more accurately, and in a smallerfootprint construction, as a result. To achieve the beneficial motion,the apparatus may include a fixed imaging optics stage formed of anexcitation source that produces an excitation beam. The apparatus mayfurther include a movable objective stage proximal to the sample andpositioned for providing the excitation beam onto the sample and forcapturing an emission from the sample. The movable objective stage mayinclude an optical lens apparatus and a turn reflector optically coupledto the imaging optics. Further, at least one of the optical lensapparatus and the turn reflector are movable relative to one another forscanning the sample. In some implementations, such movement is achievedduring scanning of the sample. In some implementations, such movement isachieved while moving from one sample position to another sampleposition to scan a different sample at another position. The movement inthe foregoing implementations is achieved while maintaining a fixedoptical path length between the optical lens apparatus and a fixed planein the fixed imaging optics stage, so as not to alter performance of theimaging optics stage.

In some implementations, the imaging system is movable in two orthogonaldirections, for example, to maintain a fixed optical path length forsampling beams of different wavelengths, i.e., for multi-spectralimaging. In some implementations, compensation plates are includedwithin the optical path to facilitate multi-spectral imaging. In someimplementations, compensation plates are separate, dedicated plates tocompensate for different excitation beam paths in a multi-spectralimaging example. In some implementations, compensation plates areintegrated with turn reflectors. In some implementations, compensationfor differences in sampling beam wavelength is achieved throughdifferent dedicated turn reflector assemblies, one for each excitationbeam.

FIG. 1 illustrates a schematic diagram of an example implementation ofthe techniques herein. FIG. 1 illustrates an optical imager apparatus100 that, in accordance with an example, includes an imaging system 102that includes an excitation source 104, an imaging sensor 106, andimaging optics 108. At least the imaging optics 108 is formed as a fixedimaging optics stage that does not move relative to a sample 110. Forexample, the imaging optics 108 may be in fixed position engagement witha housing, frame, or other support of the imaging system 102. In someimplementations, one or both of the excitation source 104 and theimaging sensor 106 are also part of the fixed imaging optics stage thatdoes not move relative to the sample 110. For example, the excitationsource 104, the imaging sensor 106, and the imaging optics 108 may be ina fixed position engagement with the housing, frame, or other support ofthe imaging system 102.

The excitation source 104 generates an excitation beam and may be alaser source, light emitting diode, or other illumination excitationsource. In some implementations, the excitation source 104 generates anexcitation beam having a single central wavelength. In someimplementations, the excitation sources 104 are formed of two or moreexcitation sources each producing a respective excitation at a differentwavelength. The sensor 106 receives an emission from the sample and maybe any solid-state imaging device, such as a include a charge coupleddevice (CCD) and/or a complementary metal oxide semiconductor (CMOS), orany suitable imager that may be used in fluorescence spectroscopy.

Contrasting to the imaging optics 108, in the illustratedimplementation, the optical image apparatus 100 further includes amovable objective stage 112 optically coupled to the imaging system 102.In the illustrated example, the movable objective stage 112 includes anoptical lens apparatus 114 that is proximal to the sample 110 and a turnreflector 116 optically coupling the optical lens apparatus 114 and theimaging optics 108. Unlike the imaging optics 108 which is maintained ina fixed position, the optical lens apparatus 114 is movable undercontrol of a controller 118, where that movement is controlled tomaintain a fixed optical path length between the optical lens apparatus114 and the imaging optics 108 or, more specifically, a fixed planewithin the imaging optics 108.

FIGS. 2A-2E illustrate examples of an optical lens apparatus 200, e.g.,a magnifying optical assembly, and a turn reflector 202 formed of twomirrors 204A & 204B joining to form a corner reflector. The optical lensapparatus 200 and the turn reflector 202 may form a movable objectivestage, for example. In the illustrated examples, imaging optics 206 isprovided as part of an imaging system, where that imaging optics 206 ismaintained in place on a frame 208, partially shown. As shown, in FIG.2A, a central axis of the optical lens apparatus 200 is laterally spacedfrom a central axis of the imaging optics 206 by a first spacingdistance, in this example 80 mm. The optical path between the exist face210 of the optical lens apparatus 200 and the entry face 212 of theimaging optics 206 is 160 mm. As both the optical lens apparatus 200 andthe turn reflector 202 are moved, that optical path length staysconstant as 160 mm, but the lateral position and spacing distancebetween the two changes. Indeed, in each of the examples, the opticallens apparatus 200 moves relative to the imaging optics 206, where onlyin the position of FIG. 2C is the central axis of the later aligned withthe central axis of the former. The examples of FIGS. 2A-2E show each ofthe optical lens apparatus 200 and the turn reflector 202 movingrelative to the fixed imaging optics 206 and movable along the y-axis.In some implementations, only one the optical lens apparatus 200 and theturn reflector 202 may be movable relative to the imaging optics 206.For example, the optical lens apparatus 200 may be maintained in a fixedposition relative to a sample, and only the turn reflector 202 ismovable along the y-axis. In this case, the path length is no longerconstant, but the path length change is less than it would be withoutthe turn reflector 202.

FIGS. 3 and 4 illustrate two different positions of a movable objectivestage and illustrate a constant optical path for each. An optical lensapparatus 300 has an entrance face 300A (e.g., corresponding to a firstlens element) and an exit face 300B (e.g., corresponding to a secondlens element) and is positioned to capture an emission from a sampleplane 302. A turn reflector 304 is formed of a right angle reflector 306and an exit reflector 308. In some examples, the right angle reflector306 is a prism reflector or two air-spaced mirrors. An imaging opticsstage 310 is shown optically coupled to the turn reflector 304 andformed of a tube lens 312 focusing the emission from the sample plane302 on a sensor 314, where the imaging optics 310 and sensor 314 aremaintained in a fixed position in both FIGS. 3 and 4 . As illustrated,in various implementations, the imaging optics stage 310 and the opticallens apparatus 300 and turn reflector 304 form a relay lens assembly forimaging the emission from the sample plane 302 onto the into the sensor314. In some implementations, the imaging optics stage 310 and theoptical lens apparatus 300 and turn reflector 304 form an infinite ornear-infinite conjugate lens assembly. In some implementations, theimaging optics stage 310 and the optical lens apparatus 300 with theturn reflector 304 each form a conjugate lens assembly.

FIGS. 5-7 illustrate another example apparatus 400 for implementing thetechniques herein. A movable objective assembly 402 is formed of anoptical lens apparatus 404, e.g., an objective lens barrel, mounted on amovable stage carriage 406, for suspending above a sample 408. Themovable objective assembly 402 includes a first reflector 410 positionedto receive an excitation beam from an excitation source 412, which maybe an illumination fiber or any other suitable excitation source, anddirect that excitation beam through a dichroic mirror 414 into theoptical lens apparatus 404 for scanning the sample 408. In theillustrated example, the movable objective assembly 402 includes aZ-axis stage 416 and the movable stage carriage 406 positionable along aY-axis stage 418, each controlled by a controller 420 to move themovable objective assembly 402 along the respective axes. FIG. 5illustrates the apparatus 400 in a first position for scanning thesample 408 at a first position. FIGS. 6 and 7 illustrate the apparatus400 in second and third positions for scanning samples 422 and 424 atrespective second and third positions.

In addition to the movable objective assembly 402, a turn reflector 426is mounted to a movable stage carriage 428 positionable along the Y-axisstage 418 under control of the controller 420, where during operationthe turn reflector 426 receives an emission beam 430 of the sample fromthe dichroic mirror 414 and provides that emission beam 430 to a fixedimaging optics stage 432 for capturing at a sensor 434. The movableobjective assembly 402 and the turn reflector 426, and their respectivecarriages and moving assemblies form a movable objective stage. In eachof the positions in FIGS. 5-7 , stages 406 and 428 have been movedrelative to one another, while the imaging optics stage 432 has remainedfixed, to maintain a constant optical path length. The movable stages406 and 428 may deploy servo controls to control operation. In otherembodiments, the movable carriage 428 may be positioned along a separatestage parallel to Y-axis stage 418.

While not shown, the controller 420 (or any of the controllers describedand/or illustrated herein) may include one or more processors and one ormore computer readable memories storing instructions that may beexecuted by the one or more processors to perform various functionsincluding the disclosed implementation. The controller 420 may include auser interface and a communication interface, electrically and/orcommunicatively coupled to the one or more processors, as are the one ormore memories.

In an implementation, the user interface may be adapted to receive inputfrom a user and to provide information to the user associated with theoperation of the apparatus 400. The user interface may include a touchscreen, a display, a keyboard, a speaker(s), a mouse, a track ball,and/or a voice recognition system. The touch screen and/or the displaymay display a graphical user interface (GUI).

In an implementation, a communication interface is adapted to enablecommunication between the apparatus 400 and a remote system(s) (e.g.,computers) via a network(s). The network(s) may include the Internet, anintranet, a local-area network (LAN), a wide-area network (WAN), acoaxial-cable network, a wireless network, a wired network, a satellitenetwork, a digital subscriber line (DSL) network, a cellular network, aBluetooth connection, a near field communication (NFC) connection, etc.Some of the communications provided to the remote system may beassociated with analysis results, imaging data, etc. generated orotherwise obtained by the apparatus 400.

The one or more processors of the controller 420 may include one or moreof a processor-based system(s) or a microprocessor-based system(s). Insome implementations, the one or more processors includes one or more ofa programmable processor, a programmable controller, a microprocessor, amicrocontroller, a graphics processing unit (GPU), a digital signalprocessor (DSP), a reduced-instruction set computer (RISC), anapplication specific integrated circuit (ASIC), a field programmablegate array (FPGA), a field programmable logic device (FPLD), a logiccircuit, and/or another logic-based device executing various functionsincluding the ones described herein.

The one or more memories can include one or more of a semiconductormemory, a magnetically readable memory, an optical memory, a hard diskdrive (HDD), an optical storage drive, a solid-state storage device, asolid-state drive (SSD), a flash memory, a read-only memory (ROM),erasable programmable read-only memory (EPROM), electrically erasableprogrammable read-only memory (EEPROM), a random-access memory (RAM), anon-volatile RAM (NVRAM) memory, a compact disc (CD), a compact discread-only memory (CD-ROM), a digital versatile disk (DVD), a Blu-raydisk, a redundant array of independent disks (RAID) system, a cache,and/or any other storage device or storage disk in which information isstored for any duration (e.g., permanently, temporarily, for extendedperiods of time, for buffering, for caching).

FIG. 8 illustrates an example method 500 of operation of the apparatus400. At a block 502 the controller 420 controls the Y-axis position ofthe movable stage 406 to align the optical lens apparatus 404 with thesample 408 for scanning. At block 504, an excitation beam from theexcitation source 412 is provided to the sample 408 and an emission beamis captured by the optical lens assembly 404 and provided to the sensor434. In an example implementation, optional image processing isperformed at a block 506 on image data from the sensor 434 to determineif the sample is in sufficient focus, i.e., if the Z-axis distancebetween the optical lens assembly 404 and the sample is within a rangeof acceptable imaging quality. If it is determined that an adjustment tothe distance is needed, at optional block 507, the controller 420controls the Z-stage 416 to adjust the distance until an acceptableimaging quality is achieved. In some examples, the block 507 isperformed to achieve an initial desired image quality. In some examples,the block 507 may be used to correct for vibrational effects or otheranomalies that affect image quality during emission capture. In someexamples, at the block 507, the controller 420 may adjust the turnreflector, e.g., stage 428, to achieve a desired initial image qualityand/or to compensate for vibrational effects. In some examples, thecontroller 420 may adjust both the Z-stage 416 and one or both of thestages 406 and 428 to compensate for vibrational effects. In someexamples, the controller 420 may adjust one or both of the stages 406and 428 to compensate for vibrational effects.

With the Z-axis position of the movable objective stage 402 established,at a block 508, the image data is assessed to determine if an adjustmentto the optical path length from the sample 408 to the sensor 434 isneeded. For example, the optical path length may change from a desiredvalue, if the apparatus 400 experiences optical jitter or if there isdrift of one of the movable stages or if the sample has moved, or due toother anomalies. In response, at a block 510, the controller adjusts oneof the movable stage 406 and or 428 to correct for the change in theoptical path length. For example, the controller 420 may control themovable stage 428 to move the turn reflector 426 relative to the opticallens apparatus 404 to correct for changes in the optical path lengthduring scanning of the sample 408.

Once the sample 408 has been scanned, at a block 512, the controller 420controls one or both of the movable stages 406 and 428 to move one orboth of the optical lens apparatus 404 or turn reflector 426,respectively, to reposition the apparatus 400 to scan the sample 422,while maintaining the optical path length established during scanning ofthe sample 408. In some implementations, the controller 420 ensures theoptical path length is fixed throughout the movement of the apparatusfrom the position in FIG. 5 to that of FIG. 6 and to that of FIG. 7 ,for example. For example, the controller 420 may continuously move thestages 406 and 428, as the apparatus 400 is moved between differentscanning positions (e.g., corresponding to positions of samples 408,422, and 424) while maintaining the optical path length fixed duringmovement. In some implementations, the optical path length need not bemaintained fixed continuously throughout the movement, but rather thecontroller 420 ensures that when the optical lens apparatus 404 iscentered for scanning the sample 408, 422, and 424, the optical pathlength at each position is the same. The process 500 then repeats withscanning of the second sample and the second position.

In various examples, the controller 420 controls movement of the stages406 and 428 at different increments. For example, the Y-axis movement ofthe stage 406 may be performed at different distance increments than theY-axis movement of the stage 428. The controller 420 may also controlthe movement along different axes at different increments, for example,controlling Z-axis movement of the stage 416 at different incrementsthan the Y-axis movement of the stage 406.

In various implementations, wavelength-dependent spatial separation maybe induced in the apparatuses herein to allow for imaging emissionsagainst two different imaging sensors, each displaced from one another.In some implementations, an optical path compensator is used toestablish optical path length matching between the two spatiallyseparated emission beams.

In various implementations, the excitation source may include multipleexcitation sources, each producing an excitation beam at a differentwavelength, and corresponding the emissions captured from the sample maybe at different wavelengths. Therefore, in some implementations, theapparatuses herein compensates for the differences in emissions anddifferent optical paths lengths experienced by the emissions, whilestill maintaining fixed optical paths lengths during sample scanning,during movement to different sampling positions, and/or during scanningat the different sampling positions. In some implementations, thismultiple-emission optical path length control is facilitated by the useof multiple imaging sensors displaced from one another.

In any of these implementations, examples of which are shown in FIGS. 9,10, and 11A-22D, compensation and/or wavelength separation may beachieved in an infinite space region of the apparatus, such as betweenan exist face 210 of the optical lens apparatus 200 and the entry face212 of the imaging optics 206. In yet other implementations of theexamples of FIGS. 9, 10, and 11A-22D, compensation and/or wavelengthseparation may be achieved in converging space, such as between theimaging optics 206 and the one or more imaging sensors 106.

FIG. 9 illustrates a turn reflector 600 formed of two angled reflectors602 and 604 forming a right-angle reflector that may be used in infinitespace or converging space. In an example, the turn reflector 600 ismoveable along a Y-axis, although the moveable stage is not shown. Tocompensate for a multiple wavelength emission beam, the first reflector602 is a dichroic designed to induce spatial separation of the emissionbeam into a first reflected wavelength beam from a first surface 602Aand a second reflected wavelength beam from a second surface 602B,generating two different beam paths 606 and 608, one for each emissionwavelength beam. In an implementation where this wavelength-basedseparation takes place after the lens 210 and before the imaging optics206, to compensate for an optical path length difference imposed by thedichroic reflector 602, a transparent optical compensation plate 610 isintroduced into the beam path 606, bring both beams into phase again. Insome examples, the optical length and material of the opticalcompensation plate 610 are determined based on the desired wavelength ofthe emission reflected at the first surface 602A and the amount of theoptical path length delay induced by the size of the spacing gap of thesurfaces 602A and 602B and the wavelength of the emission reflected atthe second surface 602B. In various implementations, the compensationplate 610 is an electro-optic compensator where the amount of opticalcompensation is controlled by signals from a controller (not shown). Invarious implementations where the wavelength-based separation takesplace in a converging space between the imaging optics 206 and the oneor more imaging sensors 106. In various implementations, thecompensation plate 610 may be a clocked compensator.

Because of the compensation plate 610, the two spatially separatedemission beam paths 606 and 608 are incident with the same optical pathentering an exit reflector 612 that couples the emission beams into thefixed imaging optics (not shown) or into spaced apart sensors 614 and616, depending on the implementation. In an example implementation inconverging space, the sensors 614 and 616 may each be configured tocapture a different emission wavelength (e.g., positioned in offsetlocations, provided with a wavelength bandpass filter, or using otherconfiguration). While the reflector 602 is shown as a dichroic, in someimplementations the reflector 604 may be a dichroic. In someimplementations both reflectors 602 and 604 may be dichroic reflectors.Further, in some implementations, the compensation plate 610 ispositioned after the reflector 604, e.g., before the exit reflector 612,to ensure that the two emission beams have the same optical path length.In some examples, an aperture may be introduced into one or both of thebeam paths 606 and 608 to prevent unwanted beam divergence and to ensurethe optical lens apparatus proximal to the sample and the fixed imagingoptics proximal to the sample form a sufficiently high-resolution relaylens configuration. For example, one or more apertures may be introducedto ensure the beam paths 606 and 608 properly coincide with an entranceaperture of the fixed imaging optics.

In some implementations, one of more of the reflectors forming a turnreflector herein is designed to reflect an emission off of a backsurface, whether as a uniform wavelength reflector or a dichroic. Insome such examples, depending upon reflector geometry and the length ofthe optical paths from the optical lens apparatus through the fixedimaging optics, such reflectors may introduce an astigmatism on theemission. Therefore, to compensate, in some examples, a tiltedcompensation plate is used in the optical beam path of the emission. Anexample configuration in shown in FIG. 10 , in which a first reflector700 reflects a single or multi-spectral) incident beam 702 off a backsurface mirror 700B (e.g., a mirror positioned on or near the backsurface of the reflector 700). A compensation plate 704 is positioned toreceive the emission and titled to correct for an astigmatism introducedby the reflector 700. For example, the compensation plate 704 may betilted an equal and opposite amount to that of the reflector 700compensating for the astigmatism within the turn reflector, e.g., beforethe emission is incident on the second turn reflector (not shown).

FIGS. 11A-11D illustrate an example turn reflector correction assembly650 that induces wavelength-dependent spatial separation of an incidentemission beam 652, which in this example results from fixed imagingoptics 654, such as the imaging optics 108 and 206, receiving theemission beam from an exit surface 656 of a movable objective stage orother upstream optical assembly (see, FIG. 11A). As shown in FIG. 11A, aturn reflector 658 has a front face 658A and a back surface mirror 658B(e.g., a mirror positioned on or near the back surface of the reflector658), each reflecting a different wavelength of incident light,generating two spatially separated beams 660 and 662, respectively. Thefirst beam 660 contains a first compensation plate 664 within the beampath between the turn reflector 658 and a first sensor 668. The secondbeam 662 contains a compensation plate assembly 670 within the beam pathbetween the turn reflector 658 and a second sensor 672. The emissiondivided into the first and second emission wavelengths may originatefrom the same position on the sample. As shown in FIG. 11B, thecompensation plate 664 is oriented substantially perpendicular to thewavefront of the beam 660. As shown in FIG. 11C, however, the beam 662confronts the compensation plate assembly 670 which, in the illustratedimplementation, is formed of two tilted compensation plates 674 and 676.The plates 674 and 676 are tilted about the Z-axis as shown and by anequal and opposite amount compensating for one another. In theillustrated example, the compensation plates 674 and 676 are clocked, inthat the turn reflector 658 is tilted about a X-axis while thecompensation plates are tilted by rotating about a Z-axis, whichcorresponds to an optical axis from the exit surface 656. The beam 662propagates along an optical axis corresponding to the Y-axis. FIG. 11Dillustrates a side cross-sectional view of the turn reflector correctionassembly 650.

To facilitate movement of the movable objective stage translatorymovement may be achieved in two directions, both along the X-axis andthe Y-axis. FIG. 12 illustrates an example configuration 800. A fixedimaging optics 802 is shown focusing an image of an emission from asample onto a sensor (not shown) at a focal position 804. A turnreflector 806 provides the emission captured by an object lens apparatus808 to the fixed imaging optics 802. In accordance with examplesdiscussed above, the turn reflector 806 is able to translate along anY-axis through movement of a Y-stage 810. The position of the objectivelens apparatus 808, however, is additionally controlled to translatealong the Y-axis and/or the X-axis via a separately controller Y-stage812 and an X-stage 814. As shown, by having two translational movementstages, the object lens apparatus 808 is able to be moved in along theX- and Y-axes to allow for scanning across a large sample area or toallow movement of the apparatus to scan samples at different positions.Further by having the translational movement stage for the turnreflector 806, the optical path length can be maintained fixed from themovable object lens apparatus 808 through the fixed imaging optics 802,even as the former is moved, by additionally translating the turnreflector 806 a sufficient amount to keep compensate for inducedshortening or lengthening of the optical path length.

FIG. 13 illustrates a schematic diagram of an implementation of a system1000 in accordance with the teachings of this disclosure. The system1000 can be used to perform an analysis on one or more samples ofinterest. The sample may include one or more DNA clusters that have beenlinearized to form a single stranded DNA (sstDNA). In the implementationshown, the system 1000 receives a reagent cartridge 1002 and includes,in part, a drive assembly 1004 and a controller 1006. The system 1000also includes, an imaging system 1012, and a waste reservoir 1014. Inother implementations, the waste reservoir 1014 may be included with thereagent cartridge 1002. The controller 1006 is electrically and/orcommunicatively coupled to the drive assembly 1004, and the imagingsystem 1012 and causes the drive assembly 1004, and/or the imagingsystem 1012 to perform various functions as disclosed herein.

The reagent cartridge 1002 carries the sample of interest that can beloaded into channels of a flow cell 1020. As used herein, a “flow cell”can include a device having a lid extending over a reaction structure toform a flow channel therebetween that is in communication with aplurality of reaction sites of the reaction structure, and can include adetection device that detects designated reactions that occur at orproximate to the reaction sites. The drive assembly 1004 interfaces withthe reagent cartridge 1002 to flow one or more reagents (e.g., A, T, G,C nucleotides) through flow cell 1020 that interact with the sample.

In an implementation, a reversible terminator is attached to the reagentto allow a single nucleotide to be incorporated onto a growing DNAstrand. In some such implementations, one or more of the nucleotides hasa unique fluorescent label that emits a color when excited. The color(or absence thereof) is used to detect the corresponding nucleotide. Inthe implementation shown, the imaging system 1012 excites one or more ofthe identifiable labels (e.g., a fluorescent label) and thereafterobtains image data for the identifiable labels. The labels may beexcited by incident light and/or a laser and the image data may includeone or more colors emitted by the respective labels in response to theexcitation. The image data (e.g., detection data) may be analyzed by thesystem 1000. The imaging system 1012 may be a fluorescencespectrophotometer including an objective lens and/or a solid-stateimaging device. The solid-state imaging device may include a chargecoupled device (CCD) and/or a complementary metal oxide semiconductor(CMOS).

After the image data is obtained, the drive assembly 1004 interfaceswith the reagent cartridge 1002 to flow another reaction component(e.g., a reagent) through the reagent cartridge 1002 that is thereafterreceived by the waste reservoir 1014 and/or otherwise exhausted by thereagent cartridge 1002. The reaction component performs a flushingoperation that chemically cleaves the fluorescent label and thereversible terminator from the sstDNA. The sstDNA is then ready foranother cycle.

Referring now to the drive assembly 1004, in the implementation shown,the drive assembly 1004 includes a pump drive assembly 1022, a valvedrive assembly 1024, and an actuator assembly 192. The pump driveassembly 1022 interfaces with a pump 1026 to pump fluid through thereagent cartridge 1002 and/or the flow cell 1020 and the valve driveassembly 1024 interfaces with a valve 1028 to control the position ofthe valve 1028. The interaction between the valve 1028 and the valvedrive assembly 1024 selectively actuates the valve 1028 to control theflow of fluid through fluidic lines 1030 of the reagent cartridge 1002.One or more of the fluidic lines 1030 fluidically couple one or morereagent reservoirs 1032 and the flow cell 1020. One or more of thevalves 1028 may be implemented by a valve manifold, a rotary valve, apinch valve, a flat valve, a solenoid valve, a reed valve, a checkvalve, a piezo valve, etc.

Referring to the controller 1006, in the implementation shown, thecontroller 1006 includes a user interface 1034, a communicationinterface 1036, one or more processors 1038, and a memory 1040 storinginstructions executable by the one or more processors 1038 to performvarious functions including the disclosed implementations. The userinterface 1034, the communication interface 1036, and the memory 1040are electrically and/or communicatively coupled to the one or moreprocessors 1038.

In an implementation, the user interface 1034 receives input from a userand provides information to the user associated with the operation ofthe system 100 and/or an analysis taking place. The user interface 1034may include a touch screen, a display, a key board, a speaker(s), amouse, a track ball, and/or a voice recognition system. The touch screenand/or the display may display a graphical user interface (GUI).

In an implementation, the communication interface 1036 enablescommunication between the system 100 and a remote system(s) (e.g.,computers) via a network(s). The network(s) may include an intranet, alocal-area network (LAN), a wide-area network (WAN), the intranet, etc.Some of the communications provided to the remote system may beassociated with analysis results, imaging data, etc. generated orotherwise obtained by the system 100. Some of the communicationsprovided to the system 100 may be associated with a fluidics analysisoperation, patient records, and/or a protocol(s) to be executed by thesystem 100.

The one or more processors 1038 and/or the system 100 may include one ormore of a processor-based system(s) or a microprocessor-based system(s).In some implementations, the one or more processors 1038 and/or thesystem 100 includes a reduced-instruction set computer(s) (RISC), anapplication specific integrated circuit(s) (ASICs), a field programmablegate array(s) (FPGAs), a field programmable logic device(s) (FPLD(s)), alogic circuit(s), and/or another logic-based device executing variousfunctions including the ones described herein.

The memory 1040 can include one or more of a hard disk drive, a flashmemory, a read-only memory (ROM), erasable programmable read-only memory(EPROM), electrically erasable programmable read-only memory (EEPROM), arandom-access memory (RAM), non-volatile RAM (NVRAM) memory, a compactdisk (CD), a digital versatile disk (DVD), a cache, and/or any otherstorage device or storage disk in which information is stored for anyduration (e.g., permanently, temporarily, for extended periods of time,for buffering, for caching).

At least one aspect of this disclosure is also directed to an apparatusand method for imaging including an imaging system that includes twoseparately movable optical stages: (i) a movable objective stageincluding an objective; and (ii) a movable imaging stage includingimaging optics and an imaging sensor. The objective stage can be movedproximal to a sample of a plurality of samples for projecting a samplingbeam onto the sample, and for capturing a fluorescence emission from thesample resulting from the sampling beam. The objective stage can includea coupler to receive the sampling beam via an optical fiber, forexample. The objective stage and the imaging stage can be moved tomaintain a substantially constant optical path length between theobjective stage and the imaging stage.

The apparatus can counter move the imaging stage relative to theobjective stage to reduce torque, rotating modes, and vibrations in theimaging system. The imaging stage can be moved opposite the objectivestage by a generally equal amount, for example. The masses of theobjective stage and the imaging stage can be matched to further reducetorque, rotating modes, and/or vibrations in the imaging system. Forcesused to move the objective stage and the imaging stage can be appliedalong centers of mass of the stages to further reduce rotational modes.

FIG. 14 is a schematic illustration of an example imaging system 1400that can be used to implement the disclosed implementations. The imagingsystem 1400 is shown including an excitation source 1402 for generatinga sampling beam 1404, a movable imaging stage 1406, a movable objectivestage 1408 including an objective 1409, a first actuator 1414 that iscontrollable for moving the objective stage 1408 between samples 1415, asecond actuator 1416 that is controllable for moving the imaging stage1406, and a controller 1417. The imaging system 1400 may be referred toas an apparatus.

The objective stage 1408 is configured to receive the sampling beam 1404from the excitation source 1402, project the sampling beam 1404 onto thesample 1415, and capture an emission from the sample 1415 resulting fromthe sampling beam 1404. The imaging stage 1406 includes an imagingsensor 1420 and imaging optics 1424 for imaging the emission from thesample 1415 onto the imaging sensor 1420. The controller 1417 isconfigured to control the first actuator 1414 and the second actuator1416 in operation such that the imaging stage 1406 moves counter to theobjective stage 1408 to allow a length of an optical path 1426 betweenthe objective 1409 and the imaging sensor 1420 to remain substantiallyconstant. The controller 1417 thus controls the excitation source 1402,the imaging stage 1406, the objective stage 1408, the first actuator1414, the second actuator 1416, and/or, more generally, the imagingsystem 1400 as a whole.

The imaging system 1400 includes coupling optics 1428 positioned betweenthe objective stage 1408 and the imaging stage 1406 along the opticalpath 1426. The coupling optics 1428 are fixed in the implementationshown and include a pair of turning mirrors 1430, 1432 positionedbetween the objective stage 1408 and the imaging stage 1406 along theoptical path 1426. The turning mirrors 1430, 1432 have faces 1434, 1436positioned at approximately 45° angles. The faces 1434, 1436 may bepositioned at another angle relative to one another, however.

The controller 1417 is configured to cause the first actuator 1414 tomove the objective stage 1408 toward the coupling optics 1428 inoperation and cause the second actuator 1416 to move the imaging stage1406 away from the coupling optics 1428. The controller 1417 is alsoconfigured to cause the first actuator 1414 to move the objective stage1408 away from the coupling optics 1428 in operation and cause thesecond actuator 1416 to move the imaging stage 1406 toward the couplingoptics 1428. The optical path 1426 can have a substantially constantoptical path length between the objective stage 1408 and the imagingstage 1406 as a result of moving the objective stage 1408 and theimaging stage 1406 in different directions.

The imaging optics 1424 of the imaging stage 1406 has relay optics 1438and the objective stage 1408 also has imaging optics 1440 includingrelay optics 1426. The relay optics 1438 of the imaging stage 1406 andthe relay optics 1442 of the objective stage 1408 focus and/or reshapethe beam to compensate for spatial dispersion between the objectivestage 1408 and the imaging stage 1406. The relay optics 1438, 1442 thuscompensate for a long optical path 1426 between the objective 1409 andthe imaging sensor 1420, in some implementations.

At least one of the first actuator 1414 or the second actuator 1416 mayinclude a drive motor, a linear motor, a voice coil motor, a ball screw,a stepper motor, or a belt drive. Other types of actuators 1414, 1416may prove suitable, however.

The excitation source 1402 can be a laser source, a light emittingdiode, or any other source of excitatory illumination useful forfluorescence spectroscopy, or other purposes. The excitation source 1402may generate the sampling beam 1404 to have a single central wavelength.The excitation source 1402 may alternatively include two or moreexcitation sources, each producing a respective excitation at adifferent wavelength.

The imaging stage 1406 can include the imaging optics 1424 having anynumber and/or type(s) of optical components for imaging or projectingemissions from the sample 1415 onto the imaging sensor 1420. The opticalcomponents may include lenses, tube lenses, apertures, mirrors, etc.

The objective stage 1408 can also include the imaging optics 1440comprising any number and/or type(s) of optical components for imagingor projecting emissions from the sample 1415 onto the imaging stage1406. Example optical components include lenses, apertures, mirrors,etc. The imaging optics 1440 can also include one or more turningmirrors 1430, 1432 for re-directing the sampling beam 1404 from an inputoptical fiber coupler (not shown for clarity of illustration) toward theobjective 1409 and the sample 1415, and/or re-directing emissions fromthe sample 1415 toward the imaging stage 1406.

The imaging sensor 1420 captures image data representing images ofemissions from the sample 1415 resulting from the sampling beam 1404.The imaging sensor 1420 can be any solid-state imaging device, such as acharge coupled device (CCD), a complementary metal oxide semiconductor(CMOS) device, or any suitable imaging sensor that can be used influorescence spectroscopy, or for other purposes.

While not shown for clarity of illustration, the controller 1417 (or anyof the controllers described and/or illustrated herein) can include oneor more processors, one or more computer-readable memories storingcomputer-readable instructions that can be executed by the one or moreprocessors to perform various functions including the disclosedimplementation, a user interface, and a communication interfaceelectrically and/or communicatively coupled to the one or moreprocessors, as are the one or more memories.

The user interface can be adapted to receive input from a user and toprovide information to the user associated with the operation of theimaging system 1400. The user interface can include a touch screen, adisplay, a keyboard, a speaker(s), a mouse, a track ball, and/or a voicerecognition system. The touch screen and/or the display can display agraphical user interface (GUI).

The communication interface can be adapted to enable communicationbetween the imaging system 1400 and a remote system(s) (e.g., computers)via one or more network(s). The network(s) can include the Internet, anintranet, a local-area network (LAN), a wide-area network (WAN), acoaxial-cable network, a wireless network, a wired network, a satellitenetwork, a digital subscriber line (DSL) network, a cellular network, aBluetooth connection, a near field communication (NFC) connection, etc.Some of the communications provided to the remote system can beassociated with analysis results, imaging data, etc. generated orotherwise obtained by the imaging system 1400.

The one or more processors of the controller 1417 can include one ormore of a processor-based system(s) or a microprocessor-based system(s).In some implementations, the one or more processors include one or moreof a programmable processor, a programmable controller, amicroprocessor, a microcontroller, a graphics processing unit (GPU), adigital signal processor (DSP), a reduced-instruction set computer(RISC), an application specific integrated circuit (ASIC), a fieldprogrammable gate array (FPGA), a field programmable logic device(FPLD), a logic circuit, and/or another logic-based device executingvarious functions including the ones described herein.

The one or more computer-readable memories can include one or more of asemiconductor memory, a magnetically readable memory, an optical memory,a hard disk drive (HDD), an optical storage drive, a solid-state storagedevice, a solid-state drive (SSD), a flash memory, a read-only memory(ROM), erasable programmable read-only memory (EPROM), electricallyerasable programmable read-only memory (EEPROM), a random-access memory(RAM), a non-volatile RAM (NVRAM) memory, a compact disc (CD), a compactdisc read-only memory (CD-ROM), a digital versatile disk (DVD), aBlu-ray disk, a redundant array of independent disks (RAID) system, acache, and/or any other storage device or storage disk in whichinformation is stored for any duration (e.g., permanently, temporarily,for extended periods of time, for buffering, for caching).

In the following examples and the claims of this patent, references aremade to axes, orientations, parallel aspects, perpendicular aspect, sameamounts, positions, proximal, etc. While such relationships can beprecise, persons of ordinary skill in the art will readily appreciatethat in practice such relationships will not, and need not be precise,but will have associated tolerances or differences. Such tolerances anddifferences can be due to, for example, manufacturing tolerances,alignment tolerances, wear, etc. Moreover, terms such as, but notlimited to, approximately, generally, substantially, etc. are usedherein to indicate that a precise value is not required, need not bespecified, etc. For example, a first value being approximately a secondvalue means that from a practical implementation perspective they can beconsidered as if equal. As used herein, such terms will have ready andinstant meaning to one of ordinary skill in the art. The terms“substantially,” “essentially,” “approximately,” “about,” “generally,”or any other version thereof, can be defined as being close to asunderstood by one of ordinary skill in the art, and in one non-limitingembodiment the term is defined to be within 10%, in another embodimentwithin 5%, in another embodiment within 1%, and in another embodimentwithin 0.5%.

FIG. 15 is a top down view of a portion of an example imaging system1500 that can be used to implement the imaging system 1400 of FIG. 14 .The imaging system 1500 includes the imaging stage 1406, the objectivestage 1408, and the coupling optics 1428. The coupling optics 1428include a pair of turn mirrors 1430, 1432. The turn mirrors 1430, 1432are fixed in the implementation shown and the imaging stage 1406 and theobjective stage 1408 move relative to the fixed mirrors and indirections generally indicated by arrows 1510, 1512.

The x-axis may be oriented right and left across the page, the y-axismay be oriented up and down the page, and the z-axis may be orientedinto and out of the page in the orientation of the illustrated exampleof FIG. 15 . The optical axis of the objective 1409 is orientedgenerally parallel to the z-axis in the implementation shown such thatthe objective 1409 can be used to image a sample 1415 beneath theimaging system 1500. The sample 1415 is beneath the page in theorientation of FIG. 15 . The objective 1409 can be moved by the actuator1414 up and down generally parallel to the y-axis in a directiongenerally indicated by arrows 1510, 1512, such that the objective 1409can be selectively positioned generally above a particular sample 1415located beneath the imaging system 1500.

The imaging optics 1440 of FIG. 15 include a mirror 1514 that redirectsemissions 1504 from a sample 1415 that are passing upward through theobjective 1409 towards the pair of turning mirrors 1430 and 1432 of thecoupling optics 1428. The turning mirrors 1430, 1432 turn the emissions1504 twice and, thus, back towards and into the imaging stage 1406, asshown.

The controller 1417 controls the actuator 1414 to move the objectivestage 1408 up away from the turning mirrors 1430, 1432 in the directiongenerally indicated by arrow 1512 and controls the actuator 1416 andcauses the imaging stage 1406 to move down towards the turning mirrors1430, 1432 in the direction generally indicated by arrow 1510 bygenerally the same amount at generally the same time. The controller1417 similarly controls the actuator 1414 to move the objective stage1408 down towards the turning mirrors 1430, 1432 and in the directiongenerally indicated by arrow 1510 and controls the actuator 1416 andcauses the imaging stage 1406 to move up away from the turning mirrors1430, 1432 in the direction generally indicated by arrow 1512 bygenerally the same amount at generally the same time. The length of theoptical path 1426 from a sample 1415 to the imaging sensor 1420 canremain substantially constant by counter moving the imaging stage 1406and the objective stage 1408 in this fashion. The illustrated example ofFIG. 2 can be implemented to have a generally net zero applied force inthe y-axis direction, which can help reduce vibrations in the y-axisdirection. However, it may experience torque, which may cause a rotatingmode.

FIG. 16 is a top down view of a portion of another example imagingsystem 1600 that can be used to implement the imaging system 1400 ofFIG. 14 . The actuators 1414, 1416 are implemented by a shaft 1606having a first threaded portion 1608 and a second threaded portion 1610,corresponding ball nuts 1612, 1614, and a motor 1615 to rotate the shaft1606. The imaging stage 1406 is shown carrying the first ball nut 1612and the objective stage 1408 is shown carrying the second ball nut 1614.While described herein as using the shaft 160 and ball nuts 1612, and1614, other methods and components may be used to maintain thepositional relationship between the imaging stage 1406 and the objectivestage 1408. For example, the imaging system 1600 may implement one ormore cables, belt drive trains, or linkage bars operatively coupled tothe imaging and objective stages 1406 and 1408 to control the relativepositions of the imaging and objective stages 1406 and 1408.

The first threaded portion 1608 has threads 1616 facing a firstdirection and the second threaded portion 1610 has threads 1618 facing asecond direction different from the first direction. The threads 1616,1618 facing different directions allows the first and second threadedportions 1608, 1610 to interact with the ball nuts 1612, 1614 and movethe ball nuts 1612, 1614 toward one another in directions generallyindicated by arrows 1620, 1622 or away from one another in directionsgenerally opposite the direction indicated by arrows 1620, 1622 when themotor 1615 rotates the shaft 1606. The illustrated example of FIG. 16can be implemented to have generally net zero applied forces and torque,which can help reduce vibrations in the y-directions and reduce rotatingmodes. Large mirrors 1430, 1432 may be used depending on the distancethe objective stage 1408 is able to be moved, however. The imagingsystem 1600 may also include the relay optics 1438, 1442 that may allowthe mirrors 1430, 1432 to be a smaller size.

The x-axis may be oriented right and left across the page, the y-axismay be oriented up and down the page, and the z-axis may be orientedinto and out of the page in the orientation of the illustrated exampleof FIG. 16 . The optical axis of the objective 1409 may be orientedgenerally parallel to the z-axis such that the objective 1409 can beused to image a sample 1415 beneath the imaging system 1600. The sample1415 is beneath the page in the orientation of FIG. 16 . The objective1409 can be moved by the actuator 1414 left and right generally parallelto the x-axis in directions generally indicated by arrows 1620, 1622,such that the objective 1409 can be selectively positioned generallyabove a particular sample 1415 located beneath the imaging system 1600.

The controller 1417 can control the actuator 1416 to counter move theimaging stage 106 right away a midline 1624 by generally the same amountat generally the same time when the controller 1417 controls theactuator 1414 to move the objective stage 1408 left away from a midline1624 of the turning mirrors 1430, 1432 during use. The controller 1417can similarly control the actuator 1416 to counter move the imagingstage 1406 left towards the midline 1624 by generally the same amount atgenerally the same time when the controller 1417 controls the actuator1414 to move the objective stage 1408 right towards the midline 1624.The length of the optical path 1426 from a sample 1415 to the imagingsensor 1420 can remain substantially constant by counter moving theimaging stage 1406 and the objective stage 1408 in this fashion, asshown.

FIG. 17 is a side view of a portion of yet another example imagingsystem 1700 that can be used to implement the imaging system 1400 ofFIG. 14 . The imaging system 1700 of FIG. 17 is similar to the imagingsystem 1600 of FIG. 16 . The objective stage 1408 of the imaging system1700 further includes second coupling optics 1704, however. The secondcoupling optics 1704 may be part of the imaging optics 1440 of theimaging system 1400 of FIG. 14 . The coupling optics 1428 includes thefirst pair of turning mirrors 1430, 1432 and the second coupling optics1704 includes a second pair of turning mirrors 1706, 1708. One of thesecond pair of turning mirrors 1708 redirects the sampling beam 1404onto the sample 1415 and the other of the second pair of turning mirrors1706 redirects emissions 1504 from the sample 1415 toward the first pairof turning mirrors 1430, 1432. The mirror 1706 thus redirects emissions1504 from a sample 1415 that are passing upward through the objective1409 towards the pair of turning mirrors 1430 and 1432 of the couplingoptics 1428. The illustrated example of FIG. 17 can be implemented tohave generally net zero applied forces and torque, which can help reducevibrations in the y-directions and reduce rotating modes. The imagingsystem 1700 can include the relay optics 1438, 1442 in someimplementations to reduce effects of a long optical path 1426 from thesample 1415 to the imaging sensor 1420.

The x-axis may be oriented right and left across the page, the y-axismay be oriented into and out of the page, and the z-axis may be orientedup and down the page in the orientation of the illustrated example ofFIG. 17 . The optical axis of the objective 1409 may be orientedupright, generally parallel to the z-axis such that the objective 1409can be used to image a sample 1415 beneath the imaging system 1700 inthe implementation shown. The objective 1409 can be moved by theactuator 1414 left and right generally parallel to the x-axis and indirections generally indicated by arrows 1620, 1622, such that theobjective 1409 can be selectively positioned generally above aparticular sample 115 located beneath the imaging system 1700.

The controller 1417 controls the actuator 1414 to move the objectivestage 1408 left and in the direction generally indicated by arrow 1620during use and the controller 1417 can control the actuator 1416 tocounter move the imaging stage 106 right and in the direction generallyindicated by arrow 1622 by generally the same amount at generally thesame time. The controller 1417 can similarly control the actuator 1416to counter move the imaging stage 106 left in the direction generallyindicated by arrow 1620 by generally the same amount at generally thesame time when the controller 1417 controls the actuator 1414 to movethe objective stage 1408 right and in the direction generally indicatedby the arrow 1622. The length of the optical path 1426 from a sample1415 to the imaging sensor 1420 can remain substantially constant bycounter moving the imaging stage 1406 and the objective stage 1408 inthis fashion, as shown.

The objective stage 1408 includes a coupler 1720 in the implementationshown to receive the sampling beam 1404 via an optical fiber 1722. Theoptical fiber 1722 is flexible to accommodate changes in distancebetween the excitation source 1402 and the objective stage 1408 tomaintain a generally constant length of the excitation optical path 1426from the excitation source 1402 to the sample 1415. While not shown inFIGS. 15 and 16 for clarity of illustration, the sampling beam 1404 canbe similarly coupled to the objective stage 1408 in the imaging systems1500 and 1600.

FIG. 18 is a side view of a portion of another example imaging system1800 that can be used to implement the imaging system 100 of FIG. 14 .The imaging system 1800 of FIG. 18 is similar to the imaging system 1700of FIG. 17 . The imaging system 1800 includes the imaging stage 1406 ina different position to fold the optical path and reduce its length andthe turning mirror 1708 is omitted, however. The coupling optics 1428includes the pair of turning mirrors 1430, 1432 and the second couplingoptics 1704 includes a second turning mirror 1706. The second turningmirror 1706 redirects the sampling beam 1404 onto the sample 1415 in theimplementation shown and redirects the emissions 1504 from the sample1415 toward the first pair of turning mirrors 1430, 1432.

The imaging systems 1500, 1600, 1700 and 1800 can be implemented suchthat forces moving the imaging stage 1406 and the objective stage 1408are directed through their centers of mass to reduce the excitation ofrotational modes that may cause an imaging system to rock on itsisolators. A first center of mass of the imaging stage 1406 and a secondcenter of mass of the objective stage 1408 move along generally a sameaxis, for example. An example axis is defined by a screw, ball screw,threaded shaft turned by a motor in some implementations, wherein thefirst and second actuators 1414, 1416 are respectively oppositelythreaded regions of the screw, ball screw, threaded shaft. Moreover, themasses of the imaging stage 1406 and the objective stage 1408 can bematched to reduce torque and/or rotational modes. The objective stage1408, the first actuator 1414, the imaging stage 1406, and the secondactuator 1416 may thus be configured and arranged such that a firstcenter of mass of the objective stage 1408 and a second center of massof the imaging stage 1406 move along substantially a same axis. Theobjective stage 1408, the first actuator 1414, the imaging stage 1406,and the second actuator 1416 may also be configured and arranged suchthat moving the objective stage 1408 and the imaging stage 1406 at asame time results in substantially no net force applied to the imagingsystem 1800.

FIG. 19 illustrates a flowchart for a method of operating any of theimaging systems 1400, 1500, 1600, 1700, 1800 disclosed herein. The orderof execution of the blocks may be changed, and/or some of the blocksdescribed may be changed, eliminated, combined and/or subdivided intomultiple blocks.

The process of FIG. 19 begins with the first actuator 1414 beingcontrolled using one or more processors to move the movable objectivestage 1408 by a first amount in a first direction to optically align theobjective 1409 of the objective stage 1408 with a sample 1415 at a firstsample position (Block 1902). The second actuator 1416 is controlledusing one or more processors to move a movable imaging stage 1406 by thefirst amount in a second direction opposite the first direction (Block1904). The imaging stage 1406 includes the imaging sensor 1420. Theobjective stage 1408 and the imaging stage 1406 may be moved by thefirst amount in opposite directions to maintain a substantially constantoptical path length between the objective 1409 and the imaging sensor1420.

Controlling the first actuator 1414 may include controlling the firstactuator 1414 to move the objective stage 1408 towards a pair of turningmirrors 1430, 1432 and controlling the second actuator 1416 may includecontrolling the second actuator 1416 to move the imaging stage 1406 awayfrom the pair of turning mirrors 1430, 1432. Controlling the firstactuator 1414 may alternatively include controlling the first actuator1414 to move the objective stage 1408 away from the pair of turningmirrors 1430, 1432 and controlling the second actuator 1416 may includecontrolling the second actuator 1416 to move the imaging stage 1406toward the pair of turning mirrors 1430, 1432.

The first actuator 1414 and the second actuator 1416 may include a shaft1606 having a first threaded portion 1608 and a second threaded portion1610, corresponding first and second ball nuts 1612, 1614, and a motor1615 to rotate the shaft 1606 and controlling the first and secondactuators 1414, 1416 may include controlling the motor 1615 to rotatethe shaft 1606 such that the objective stage 1408 moves in the firstdirection, and the imaging stage 1406 moves in the second direction.Controlling the first actuator 1414 in such implementations may includecontrolling the first actuator 1414 to move the objective stage 1408towards a midline 1624 of the pair of turning mirrors 1430, 1432 andcontrolling the second actuator 1416 may include controlling the secondactuator 1416 to move the imaging stage 1406 away from the midline 1624of the pair of turning mirrors 1430, 1432.

The sampling beam 1404 is provided to the objective stage 1408 (Block1906). The objective stage 1408 is configured to project the samplingbeam 1404 onto the sample 1415. A fluorescence emission from the sample1415 resulting from the sampling beam 1404 onto the imaging sensor 1420is imaged using the objective stage 1408 and the pair of turning mirrors1430, 1432 (Block 1908). The method 1900 can be repeated to analyzeother samples 1415.

FIG. 20 illustrates a schematic diagram of an implementation of anexample system 2000 in accordance with the teachings of this disclosure.The system 2000 can be used to perform an analysis on one or moresamples of interest. The sample can include one or more DNA clustersthat have been linearized to form a single stranded DNA (sstDNA). In theimplementation shown, the system 2000 receives a reagent cartridge 2002and includes, in part, a drive assembly 2004 and a controller 2006. Thesystem 2000 also includes an imaging system 2012 and a waste reservoir2014. In other implementations, the waste reservoir 2014 can be includedwith the reagent cartridge 2002. The controller 2006 is electricallyand/or communicatively coupled to the drive assembly 2004 and theimaging system 2012, and causes the drive assembly 2004 and/or theimaging system 2012 to perform various functions as disclosed herein.

The reagent cartridge 2002 carries the sample of interest that can beloaded into channels of a flow cell 2020. As used herein, a “flow cell”can include a device having a lid extending over a reaction structure toform a flow channel therebetween that is in communication with aplurality of reaction sites of the reaction structure, and can include adetection device that detects designated reactions that occur at orproximate to the reaction sites. The drive assembly 2004 interfaces withthe reagent cartridge 2002 to flow one or more reagents (e.g., A, T, G,C nucleotides) through the flow cell 2020 that interact with the sample.

In an implementation, a reversible terminator is attached to the reagentto allow a single nucleotide to be incorporated onto a growing DNAstrand. In some such implementations, one or more of the nucleotides hasa unique fluorescent label that emits a color when excited. The color(or absence thereof) is used to detect the corresponding nucleotide. Inthe implementation shown, the imaging system 2012 excites one or more ofthe identifiable labels (e.g., a fluorescent label) and thereafterobtains image data for the identifiable labels. The labels can beexcited by incident light and/or a laser and the image data can includeone or more colors emitted by the respective labels in response to theexcitation. The image data (e.g., detection data) can be analyzed by thesystem 2000. The imaging system 2012 can be a fluorescencespectrophotometer including an objective lens and/or a solid-stateimaging device. The solid-state imaging device can include a CCD and/ora CMOS device. Example imaging systems 1400, 1500, 1600, 1700 and 1800that can be used to implement the imaging system 2012 are describedabove in connection with FIGS. 14-19 .

After the image data is obtained, the drive assembly 2004 interfaceswith the reagent cartridge 2002 to flow another reaction component(e.g., a reagent) through the reagent cartridge 2002 that is thereafterreceived by the waste reservoir 2014 and/or otherwise exhausted by thereagent cartridge 2002. The reaction component performs a flushingoperation that chemically cleaves the fluorescent label and thereversible terminator from the sstDNA. The sstDNA is then ready foranother cycle.

Referring now to the drive assembly 2004, in the implementation shown,the drive assembly 2004 includes a pump drive assembly 2022, a valvedrive assembly 2024, and an actuator assembly 192. The pump driveassembly 2022 interfaces with a pump 2026 to pump fluid through thereagent cartridge 2002 and/or the flow cell 2020 and the valve driveassembly 2024 interfaces with a valve 2028 to control the position ofthe valve 2028. The interaction between the valve 2028 and the valvedrive assembly 2024 selectively actuates the valve 2028 to control theflow of fluid through fluidic lines 2030 of the reagent cartridge 2002.One or more of the fluidic lines 2030 fluidically couple one or morereagent reservoirs 2032 and the flow cell 2020. One or more of thevalves 2028 can be implemented by a valve manifold, a rotary valve, apinch valve, a flat valve, a solenoid valve, a reed valve, a checkvalve, a piezo valve, etc.

Referring to the controller 2006, in the implementation shown, thecontroller 2006 includes a user interface 2034, a communicationinterface 2036, one or more processors 2038, and computer-readablememory 2040 storing instructions executable by the one or moreprocessors 2038 to perform various functions including the disclosedimplementations. The user interface 2034, the communication interface2036, and the memory 2040 are electrically and/or communicativelycoupled to the one or more processors 2038.

In an implementation, the user interface 2034 receives input from a userand provides information to the user associated with the operation ofthe system 2000 and/or an analysis taking place. The user interface 2034can include a touch screen, a display, a key board, a speaker(s), amouse, a track ball, and/or a voice recognition system. The touch screenand/or the display can display a graphical user interface (GUI).

In an implementation, the communication interface 2036 enablescommunication between the system 2000 and a remote system(s) (e.g.,computers) via a network(s). The network(s) can include an intranet, aLAN, a WAN, the intranet, etc. Some of the communications provided tothe remote system can be associated with analysis results, imaging data,etc. generated or otherwise obtained by the system 2000. Some of thecommunications provided to the system 2000 can be associated with afluidics analysis operation, patient records, and/or a protocol(s) to beexecuted by the system 2000.

The one or more processors 2038 and/or the system 2000 can include oneor more of a processor-based system(s) or a microprocessor-basedsystem(s). In some implementations, the one or more processors 2038and/or the system 2000 includes a RISC, an ASIC, an FPGA, an FPLD, alogic circuit, and/or another logic-based device executing variousfunctions including the ones described herein.

The memory 2040 can include one or more of a hard disk drive, a flashmemory, a ROM, an EPROM, an EEPROM, a RAM, an NVRAM, a CD, a DVD, acache, and/or any other storage device or storage disk in whichinformation is stored for any duration (e.g., permanently, temporarily,for extended periods of time, for buffering, for caching).

An apparatus comprises: an imaging system having an excitation sourcefor generating an excitation beam, a fixed imaging optics stage formedcomposed of an excitation source for generating an excitation beam, asensor for measuring an emission from a sample, and an imaging opticsfor imaging the emission from the sample onto the sensor; and a movableobjective stage proximal to the sample and positioned for providing theexcitation beam onto the sample and for capturing the emission from thesample, where the movable objective stage includes an optical lensapparatus and a turn reflector optically coupled to the imaging opticsof the fixed imaging optics stage, and where at least one of the opticallens apparatus and the turn reflector of the movable objective stage aremovable relative to one another for scanning of the sample , whilemaintaining a fixed optical path length between the optical lensapparatus and a fixed plane in the fixed imaging optics stage duringmovement.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, wherein themovable objective is movable in two orthogonal directions to maintain afixed optical path length.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, whereinexcitation source comprises a first excitation source producing a firstexcitation at a first sampling wavelength that elicits a first sampleemission range of wavelengths and a second excitation source producing asecond excitation at a second sampling wavelength that elicits a secondsample emission range of wavelengths, each of the first excitation,first emission, second excitation and second emission having arespective optical path.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, furthercomprising: a compensation plate positioned in one of the respectiveoptical paths.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, furthercomprising: a compensation plate positioned in a plurality of therespective optical paths.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, wherein both theoptical lens apparatus and the turn reflector of the movable objectivestage are movable relative to one another for scanning of a sample area.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, wherein at leastone of the optical lens apparatus and the turn reflector of the movableobjective stage are movable relative to one another for scanningmultiple samples areas at different positions.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, furthercomprising: a controller configured to move the at least one of theoptical lens apparatus and the turn reflector of the movable objectivestage while maintaining the fixed optical path length to sample at thedifferent positions.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, wherein thecontroller is configured to continuously move the optical lens apparatusand the turn reflector of the movable objective between the differentpositions.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, furthercomprising: a controller configured to continuously control movement ofthe turn reflector during capture of the emission beam from the sampleto compensate for vibrational effects during capture.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, furthercomprising: a controller configured to continuously control movement ofthe optical lens apparatus and the turn reflector during capture of theemission beam from the sample to compensate for vibrational effectsduring capture.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, wherein thecontroller is configured to continuously control movement of the opticallens apparatus and the turn reflector at different movement increments.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, furthercomprising: a controller configured to move the movable objective toachieve the fixed optical path length at each of the different samplepositions.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, furthercomprising: a z-stage adjustment controller to adjust a distance betweenthe optical lens apparatus and the sample

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, wherein thefixed imaging optics stage, the optical lens apparatus, and the turnreflector form a relay lens assembly for imaging the emission into thesensor.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, wherein thefixed imaging optics stage, the optical lens apparatus, and the turnreflector form an infinite conjugate lens assembly or near infiniteconjugate lens assembly.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, wherein thefixed imaging optics stage and the optical lens apparatus with the turnreflector each form a finite conjugate lens assembly.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, furthercomprising: one or more color separating elements between the objectiveand the fixed imaging optics to direct light of a first emissionwavelength to a first image sensor and light of a second emissionwavelength to a second image sensor.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, furthercomprising: one or more color separating elements within or after thefixed imaging optics are to direct light of a first emission wavelengthto a first image sensor and light of a second emission wavelength to asecond image sensor.

The apparatus of any one of the preceding implementations and/or any oneor more of the implementations disclosed below, wherein the movableobjective stage is separately movable along two orthogonal axes eachsubstantially planar to the sample.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, furthercomprising: a compensating plate disposed before a first image sensor.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, furthercomprising: a plurality of compensating plates disposed before a firstimage sensor.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, furthercomprising: a plurality of compensating plates disposed before a firstimage sensor and a different compensation plate or a different pluralityof compensating plates disposed before a second image sensor

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, wherein one ormore compensating plates is tilted or wedged.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, wherein themovable objective stage is separately movable along two orthogonal axeseach substantially planar to the sample.

An apparatus comprising: an imaging system having an excitation sourcefor generating an excitation beam, a fixed imaging optics stage composedof a sensor for measuring an emission from a sample, and imaging opticsfor imaging the emission from the sample onto the sensor; and anobjective stage proximal to the sample and positioned for providing theexcitation beam onto the sample and for capturing the emission from thesample, where the objective stage includes an optical lens apparatus,wherein the imaging system comprises (i) one or more color separatingelements between the objective and the fixed imaging optics to directlight of a first emission wavelength to a first image sensor of thesensor and light of a second emission wavelength to a second imagesensor of the sensor, or (ii) the one or more color separating elementswithin or after the fixed imaging optics to direct light of the firstemission wavelength to the first image sensor and light of the secondemission wavelength to the second image sensor.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, furthercomprising: a compensating plate disposed before a first image sensor.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, furthercomprising: a plurality of compensating plates are disposed before afirst image sensor.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, wherein aplurality of compensating plates is disposed before a first image sensorand before a second image sensor.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, wherein one ormore compensating plates is tilted or wedged.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, furthercomprising: a compensating plate pair disposed within a beam pathdefined by the one or more color separating elements.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, wherein thecompensating plate pair comprises a first compensating plate tilted in afirst angular direction and a second compensating plate tilted in asecond angular direction, equal and opposite to the first angulardirection.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, wherein the oneor more color separating elements are tilted above a first axis and thefirst compensating plate and the second compensating plate are eachtilted above a second axis orthogonal to the first axis.

A computer-implemented method of optically probing a sample, comprises:aligning, using one or more processors, a movable objective stage,having an optical lens apparatus and a turn reflector optically coupledto imaging optics of a fixed imaging optics stage, to align the opticallens apparatus with the sample for probing at an optical path length;providing, using the optical lens apparatus, an excitation beam to thesample and capturing, using the optical lens apparatus, a fluorescenceemission from the sample; in response to identification of a shift infocus at the sample from the fluorescence emission, adjusting a positionof the optical lens apparatus or a position of the turn reflector tocompensate for the shift; and moving, using the one or more processors,the optical lens apparatus and the turn reflector to position theoptical lens apparatus to over a subsequent sample for probing, whilemaintaining the optical path length.

The computer-implemented method of any one or more of the precedingimplementations and/or any one or more of the implementations disclosedbelow, comprises: moving, using the one or more processors, the opticallens apparatus and the turn reflector to position the optical lensapparatus to over the subsequent sample for probing while maintainingthe optical path length throughout the movement from the sample to thesubsequent sample.

The computer-implemented method of any one or more of the precedingimplementations and/or any one or more of the implementations disclosedbelow, comprises: performing imaging processing on image data containingthe fluorescence emission; and in responding to determining the imagedata does not satisfy a focusing condition, adjusting a verticaldistance between the optical lens apparatus and the sample until theimage data satisfies the focusing condition.

The computer-implemented method of any one or more of the precedingimplementations and/or any one or more of the implementations disclosedbelow, wherein moving the optical lens apparatus and the turn reflectorto position the optical lens apparatus to over the subsequent sample forprobing, while maintaining the optical path length comprises moving theoptical lens apparatus and the turn reflector in a plane substantiallyparallel to a plane containing the sample and the subsequent sample.

An implementation of an apparatus, comprising: an excitation source forgenerating a sampling beam; a movable objective stage including anobjective, the objective stage configured to receive the sampling beamfrom the excitation source, project the sampling beam onto a sample, andcapture an emission from the sample resulting from the sampling beam; amovable imaging stage including an imaging sensor, and imaging opticsfor imaging the emission from the sample onto the imaging sensor; afirst actuator controllable to move the objective stage betweendifferent sample positions; a second actuator controllable to move theimaging stage; and a controller configured to control the first actuatorand the second actuator such that the imaging stage moves counter to theobjective stage to allow a length of an optical path between theobjective and the imaging sensor to remain substantially constant.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, furthercomprising coupling optics positioned between the objective stage andthe imaging stage along the optical path.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, wherein thecoupling optics are fixed.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, wherein thecoupling optics comprise a pair of turning mirrors positioned betweenthe objective stage and the imaging stage along the optical path.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, wherein theturning mirrors have faces positioned at approximately 45° angles.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, wherein thecontroller is configured to cause the first actuator to move theobjective stage toward the coupling optics and cause the second actuatorto move the imaging stage away from the coupling optics.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, wherein thecontroller is configured to cause the first actuator to move theobjective stage away from the coupling optics and cause the secondactuator to move the imaging stage toward the coupling optics.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, wherein theimaging optics of the imaging stage comprise relay optics.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, wherein theobjective stage comprises imaging optics comprising relay optics.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, wherein therelay optics of the imaging stage and the relay optics of the objectivestage reshape at least one of the sampling beam or emission tocompensate for spatial dispersion.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, wherein at leastone of the first actuator or the second actuator comprises a drivemotor, a linear motor, a voice coil motor, a ball screw, a steppermotor, or a belt drive.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, wherein thefirst actuator and the second actuator comprise a shaft having a firstthreaded portion and a second threaded portion, corresponding first andsecond ball nuts, and a motor to rotate the shaft, the imaging stagecarrying the first ball nut and the objective stage carrying the secondball nut.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, wherein thefirst threaded portion has threads facing a first direction and thesecond threaded portion has threads facing a second direction differentfrom the first direction.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, wherein themotor rotates the shaft in a first direction and causes the first ballnut and the second ball nut to move toward one another and wherein themotor rotates the shaft in a second direction and causes the first ballnut and the second ball nut to move away from one another.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, wherein theobjective stage further includes second coupling optics.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, wherein thecoupling optics comprise a first pair of turning mirrors and the secondcoupling optics comprise a second pair of turning mirrors.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, wherein one ofthe second pair of turning mirrors redirects the sampling beam onto thesample.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, wherein theother of the second pair of turning mirrors redirects the emissions fromthe sample toward the first pair of turning mirrors.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, wherein thecoupling optics comprise a pair of turning mirrors and the secondcoupling optics comprise a second turning mirror.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, wherein thesecond turning mirror redirects the sampling beam onto the sample.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, wherein thesecond turning mirror redirects the emissions from the sample toward thefirst pair of turning mirrors.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, wherein theobjective stage, the first actuator, the imaging stage, and the secondactuator are configured and arranged such that a first center of mass ofthe objective stage and a second center of mass of the imaging stagemove along substantially a same axis.

The apparatus of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, wherein theobjective stage, the first actuator, the imaging stage, and the secondactuator are configured and arranged such that moving the objectivestage and the imaging stage at a same time results in substantially nonet force applied to the apparatus.

An implementation of method, comprising: controlling, using one or moreprocessors, a first actuator to move a movable objective stage by afirst amount in a first direction to optically align an objective of theobjective stage with a sample at a first sample position; controlling,using one or more processors, a second actuator to move a movableimaging stage by the first amount in a second direction opposite thefirst direction, wherein the imaging stage includes an imaging sensor,and moving the objective stage and the imaging stage by the first amountin opposite directions maintains a substantially constant optical pathlength between the objective and the imaging sensor; providing asampling beam to the objective stage, the objective stage configured toproject the sampling beam onto the sample; and imaging, using theobjective stage and a pair of turning mirrors, a fluorescence emissionfrom the sample resulting from the sampling beam onto the imagingsensor.

The method of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, whereincontrolling the first actuator includes controlling the first actuatorto move the objective stage towards a pair of turning mirrors, andwherein controlling the second actuator includes controlling the secondactuator to move the imaging stage away from the pair of turningmirrors.

The method of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, whereincontrolling the first actuator includes controlling the first actuatorto move the objective stage towards a midline of the pair of turningmirrors, and wherein controlling the second actuator includescontrolling the second actuator to move the imaging stage away from themidline of the pair of turning mirrors.

The method of any one or more of the preceding implementations and/orany one or more of the implementations disclosed below, wherein thefirst actuator and the second actuator comprise a shaft having a firstthreaded portion and a second threaded portion, corresponding first andsecond ball nuts, and a motor to rotate the shaft and whereincontrolling the first and second actuators includes controlling themotor to rotate the shaft such that the objective stage moves in thefirst direction, and the imaging stage moves in the second direction.

The foregoing description is provided to enable a person skilled in theart to practice the various configurations described herein. While thesubject technology has been particularly described with reference to thevarious figures and configurations, it should be understood that theseare for illustration purposes only and should not be taken as limitingthe scope of the subject technology.

As used herein, an element or step recited in the singular and proceededwith the word “a” or “an” should be understood as not excluding pluralof said elements or steps, unless such exclusion is explicitly stated.Furthermore, references to “one implementation” are not intended to beinterpreted as excluding the existence of additional implementationsthat also incorporate the recited features. Moreover, unless explicitlystated to the contrary, implementations “comprising,” “including,” or“having” an element or a plurality of elements having a particularproperty may include additional elements whether or not they have thatproperty. Moreover, the terms “comprising,” including,” having,” or thelike are interchangeably used herein.

The terms “substantially,” “approximately,” and “about” used throughoutthis Specification are used to describe and account for smallfluctuations, such as due to variations in processing. For example, theycan refer to less than or equal to ±5%, such as less than or equal to±2%, such as less than or equal to ±1%, such as less than or equal to±0.5%, such as less than or equal to ±0.2%, such as less than or equalto ±0.1%, such as less than or equal to ±0.05%.

There may be many other ways to implement the subject technology.Various functions and elements described herein may be partitioneddifferently from those shown without departing from the scope of thesubject technology. Various modifications to these implementations maybe readily apparent to those skilled in the art, and generic principlesdefined herein may be applied to other implementations. Thus, manychanges and modifications may be made to the subject technology, by onehaving ordinary skill in the art, without departing from the scope ofthe subject technology. For instance, different numbers of a givenmodule or unit may be employed, a different type or types of a givenmodule or unit may be employed, a given module or unit may be added, ora given module or unit may be omitted.

Underlined and/or italicized headings and subheadings are used forconvenience only, do not limit the subject technology, and are notreferred to in connection with the interpretation of the description ofthe subject technology. All structural and functional equivalents to theelements of the various implementations described throughout thisdisclosure that are known or later come to be known to those of ordinaryskill in the art are expressly incorporated herein by reference andintended to be encompassed by the subject technology. Moreover, nothingdisclosed herein is intended to be dedicated to the public regardless ofwhether such disclosure is explicitly recited in the above description.

It should be appreciated that all combinations of the foregoing conceptsand additional concepts discussed in greater detail below (provided suchconcepts are not mutually inconsistent) are contemplated as being partof the subject matter disclosed herein. In particular, all combinationsof claimed subject matter appearing at the end of this disclosure arecontemplated as being part of the subject matter disclosed herein.

1-37. (canceled)
 38. An apparatus, comprising: an excitation source forgenerating a sampling beam; a movable objective stage including anobjective, the objective stage configured to receive the sampling beamfrom the excitation source, project the sampling beam onto a sample, andcapture an emission from the sample resulting from the sampling beam; amovable imaging stage including an imaging sensor, and imaging opticsfor imaging the emission from the sample onto the imaging sensor; afirst actuator controllable to move the objective stage betweendifferent sample positions; a second actuator controllable to move theimaging stage; and a controller configured to control the first actuatorand the second actuator such that the imaging stage moves counter to theobjective stage to allow a length of an optical path between theobjective and the imaging sensor to remain substantially constant. 39.The apparatus of claim 38, further comprising coupling optics positionedbetween the objective stage and the imaging stage along the opticalpath.
 40. The apparatus of claim 39, wherein the coupling optics arefixed.
 41. The apparatus of claim 39, wherein the coupling opticscomprise a pair of turning mirrors positioned between the objectivestage and the imaging stage along the optical path.
 42. The apparatus ofclaim 41, wherein the turning mirrors have faces positioned atapproximately 45° angles.
 43. The apparatus of claim 39, wherein thecontroller is configured to cause the first actuator to move theobjective stage toward the coupling optics and cause the second actuatorto move the imaging stage away from the coupling optics.
 44. Theapparatus of claim 39, wherein the controller is configured to cause thefirst actuator to move the objective stage away from the coupling opticsand cause the second actuator to move the imaging stage toward thecoupling optics.
 45. The apparatus of claim 38, wherein the imagingoptics of the imaging stage comprise relay optics.
 46. The apparatus ofclaim 38, wherein the objective stage comprises imaging opticscomprising relay optics.
 47. The apparatus of claim 45, wherein therelay optics of the imaging stage and the relay optics of the objectivestage reshape at least one of the sampling beam or emission tocompensate for spatial dispersion.
 48. The apparatus of claim 38,wherein at least one of the first actuator or the second actuatorcomprises a drive motor, a linear motor, a voice coil motor, a ballscrew, a stepper motor, or a belt drive.
 49. The apparatus of claim 38,wherein the first actuator and the second actuator comprise a shafthaving a first threaded portion and a second threaded portion,corresponding first and second ball nuts, and a motor to rotate theshaft, the imaging stage carrying the first ball nut and the objectivestage carrying the second ball nut.
 50. The apparatus of claim 49,wherein the first threaded portion has threads facing a first directionand the second threaded portion has threads facing a second directiondifferent from the first direction.
 51. The apparatus of claim 49,wherein the motor rotates the shaft in a first direction and causes thefirst ball nut and the second ball nut to move toward one another andwherein the motor rotates the shaft in a second direction and causes thefirst ball nut and the second ball nut to move away from one another.52. The apparatus of claim 39, wherein the objective stage furtherincludes second coupling optics.
 53. The apparatus of claim 52, whereinthe coupling optics comprise a first pair of turning mirrors and thesecond coupling optics comprise a second pair of turning mirrors. 54.The apparatus of claim 53, wherein one of the second pair of turningmirrors redirects the sampling beam onto the sample.
 55. The apparatusof claim 53, wherein the other of the second pair of turning mirrorsredirects the emissions from the sample toward the first pair of turningmirrors.
 56. The apparatus of claim 52, wherein the coupling opticscomprise a pair of turning mirrors and the second coupling opticscomprise a second turning mirror.
 57. The apparatus of claim 56, whereinthe second turning mirror redirects the sampling beam onto the sample.58. The apparatus of claim 56, wherein the second turning mirrorredirects the emissions from the sample toward the first pair of turningmirrors.
 59. The apparatus of claim 38, wherein the objective stage, thefirst actuator, the imaging stage, and the second actuator areconfigured and arranged such that a first center of mass of theobjective stage and a second center of mass of the imaging stage movealong substantially a same axis.
 60. The apparatus of claim 38, whereinthe objective stage, the first actuator, the imaging stage, and thesecond actuator are configured and arranged such that moving theobjective stage and the imaging stage at a same time results insubstantially no net force applied to the apparatus.
 61. A method,comprising: controlling, using one or more processors, a first actuatorto move a movable objective stage by a first amount in a first directionto optically align an objective of the objective stage with a sample ata first sample position; controlling, using one or more processors, asecond actuator to move a movable imaging stage by the first amount in asecond direction opposite the first direction, wherein the imaging stageincludes an imaging sensor, and moving the objective stage and theimaging stage by the first amount in opposite directions maintains asubstantially constant optical path length between the objective and theimaging sensor; providing a sampling beam to the objective stage, theobjective stage configured to project the sampling beam onto the sample;and imaging, using the objective stage and a pair of turning mirrors, afluorescence emission from the sample resulting from the sampling beamonto the imaging sensor.
 62. The method of claim 61, wherein controllingthe first actuator includes controlling the first actuator to move theobjective stage towards a pair of turning mirrors, and whereincontrolling the second actuator includes controlling the second actuatorto move the imaging stage away from the pair of turning mirrors.
 63. Themethod of claim 61, wherein controlling the first actuator includescontrolling the first actuator to move the objective stage towards amidline of the pair of turning mirrors, and wherein controlling thesecond actuator includes controlling the second actuator to move theimaging stage away from the midline of the pair of turning mirrors. 64.The method of claim 61, wherein the first actuator and the secondactuator comprise a shaft having a first threaded portion and a secondthreaded portion, corresponding first and second ball nuts, and a motorto rotate the shaft and wherein controlling the first and secondactuators includes controlling the motor to rotate the shaft such thatthe objective stage moves in the first direction, and the imaging stagemoves in the second direction.